Hybrid And Chimeric Polypeptides That Regulate Activation Of Complement

ABSTRACT

A hybrid complement-regulating protein comprises a first functional unit of a first complement regulatory protein having complement regulating properties, a first spacer sequence of at least about 200 amino acids encoding a polypeptide that does not exhibit complement regulating properties and at least a second functional unit attached to the spacer sequence. The second functional unit may be a polypeptide providing a functional unit of a second complement regulatory protein, a polypeptide derived from an immunoglobulin, or a polypeptide that enhances binding of the protein to an animal cell. The hybrid protein may also contain a second spacer sequence and a third functional unit of a complement regulatory protein, a polypeptide derived from an immunoglobulin, and a polypeptide that enhances binding of the protein to an animal cell. The optional third functional unit may be the same or different from the first or second functional units.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made, at least in part, with support from NIHgrant #AI23598. The U.S. government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The complement system comprises a number of serum proteins that functionin the body's immune response to infection and tissue injury. Activationof complement can occur via three pathways, the classical pathwayinvolving the binding of complement component C1q to antigen-antibodycomplexes, the lectin pathway involving binding of mannose bindinglectins to antigens, and the alternative pathway involving binding ofcomplement component C3b to an activator surface such as cell wallpolysaccharides of yeast and bacterial microorganisms. Activation ofcomplement results in the formation of anaphylatoxins (C3a and C5a),membrane attack complexes (C5b-9), and opsonins (C3b and C4b) thatamplify inflammation and destroy foreign and necrotic cells.

Complement activation is regulated by a number of plasma and cellassociated proteins. Such proteins inactivate specific steps of theclassical, lectin, and/or alternative pathway by regulating the activityof C3/C5 convertases or serving as a cofactor for the factor I cleavageof C3b and/or C4b. These proteins are either soluble plasma proteins ormembrane proteins (integral or lipid-anchored) expressed on a variety ofcell types. These proteins possess many structural similarities.

Decay Accelerating Factor (DAF)

Decay accelerating factor (DAF, CD55) is a membrane-associatedregulatory protein that protects self cells from activation ofautologous complement on their surfaces. DAF acts by rapidlydissociating C3 and C5 convertases, the central enzymes of the cascade.DAF possesses the most potent decay accelerating activity of theproteins associated with complement regulation, and acts on both theclassical pathway (C4b2a and C4b2a3b) and alternative pathway (C3bBb andC3bBbC3b) enzymes. DAF, however, does not have cofactor function.

Structural analyses of DAF have shown that, starting from itsN-terminus, it is composed of four ˜60 amino acid-long units followed bya heavily O-glycosylated serine (S) and threonine (T) rich stretch,which is, in turn, linked to a posttranslationally-addedglycoinositolphospholipid (GPI) anchor. The amino acid sequence of DAFis shown in FIG. 1 A (SEQ. ID NO: 1). The four 60 amino acid longrepeating units are termed complement control protein repeats (CCPs) orshort consensus repeats (SCRs). CCPI includes amino acids 35-95 of SEQ.ID NO: 1. CCP2 includes amino acids 97-159; CCP3 includes amino acids162-221 and CCP includes amino acids 224-284 of SEQ. ID NO: 1. Theyprovide for all of DAF's regulatory activity. The heavily O-glycosylatedregion serves as a cushion which positions the CCPs at an appropriatedistance above the surface membrane. The GPI anchor allows DAF to movefreely in the plane of the plasma membrane enabling it to inactivateconvertase complexes wherever they assemble.

The critical role that DAF plays in inhibiting complement activation isevident both from natural disease and studies in animal models employingDaf knockout mice. In the human disease paroxysmal nocturnalhemoglobinuria (PNH), mutation in the GPI anchor pathway leading to theabsence of DAF renders affected blood cells susceptible to heightenedC3b uptake and intravascular hemolysis. In the animal disease modelsemploying the Daf knockout, the absence of DAF renders the mice markedlymore susceptible to tissue damage in 1) nephrotoxic serum (NTS) inducednephritis, a model of human membranous glomerulonephritis, 2) dextransodium sulfate (DSS) induced colitis, a model of inflammatory boweldisease, and 3) anti-acetylcholine receptor (anti-AChR) inducedmyasthenia gravis, a close model of the human autoimmune disorder.

The nucleotide sequence of a cDNA encoding DAF is shown in FIG. 1B (SEQ.ID NO: 2).

Complement Receptor 1 (CR1)

Complement receptor 1 (CR1 or the C3b receptor, CD35) is another potentregulator of complement activation. Unlike DAF which functionsintrinsically to protect the cells that express it, CR1 functionsextrinsically on targets of complement attack, e.g. pathogens. CR1 is alarger molecule in that, rather than 4 CCPs, it is comprised of 30 CCPsarranged in 4 groups of 7 CCPs termed long homologous repeats (LHRs).The CCPs and LHRs of CR1 are provided in Table I below. The amino acidresidue numbers refer to the amino acid sequence provided in FIG. 2(SEQ. ID NO: 3). Functional analyses have shown that CR1 possesses bothdecay accelerating activity and cofactor activity for cleavage of C4band C3b by the serum enzyme, factor I. Early studies showed that amongcomplement regulators, it is the most potent in this latter activity andthat it is the only regulator that promotes both initial cleavage of C3bto iC3b and subsequent cleavage of the iC3b intermediate to C3dg, thesurface-bound C3b end product.

Structure-function studies of CR1 have shown that its regulatoryactivity resides primarily in its three N-terminal LHRs, i.e., LHRs A,B, and C. Functional activity within each 7 CCP LHR is containedessentially in each case in the first 3 CCPs. Recent studies have shownthat CR1's potent cofactor activity resides in LHRs B and C, while itsdecay accelerating activity resides in LHR A.

The nucleotide sequence of a cDNA encoding CR1 is shown in FIG. 3 (SEQ.ID NO: 4).

TABLE 1 Amino Acid No. Domain 1 or 6-46 Leader peptide  47-106 CCP1,begin LHR-A 107-168 CCP2 169-238 CCP3 239-300 CCP4 301-360 CCP4 361-423CCP5 424-496 CCP7, end LHR-A 497-556 CCP8, begin LHR-B 557-618 CCP9619-688 CCP10 689-750 CCP11 751-810 CCP12 811-873 CCP12 874-946 CCP14,end LHR-B  947-1006 CCP15, begin LHR-C 1007-1068 CCP15 1069-1138 CCP161139-1200 CCP17 1201-1260 CCP18 1261-1323 CCP20 1324-1399 CCP21, endLHR-C 1400-1459 CCP22, begin LHR-D 1460-1521 CCP23 1522-1591 CCP241592-1653 CCP25 1654-1713 CCP26 1714-1776 CCP27 1777-1851 CCP28, andLHR-D 1852-1911 CCP29 1912-1972 CCP30

Membrane Cofactor Protein (MCP)

MCP (also known as ‘CD46’) is present on the cell surface of a number ofcell types including peripheral blood cells (excluding erythrocytes),cells of epithelial, endothelial and fibroblast lineages, trophoblastsand sperm. MCP has four CCPs and a serine/threonine enriched region inwhich heavy O-linked glycosylation occurs. MCP also has a transmembraneand cytoplasmic domain. The structure of MCP is provided in Table 2below with reference to the amino acid sequence of MCP provided in FIG.4A (SEQ. ID NO: 5). MCP works by binding to the C3b and C4b present onthe cell surface thereby targeting C3b and C4b for degradation by factorI, a plasma protease, and thereby destroying any subsequent C3 or C4convertase activity. Thus, MCP is said to have “cofactor activity”.Because MCP is localized on the cell surface, it protects only the cellson which it is present and is therefore said to act in an intrinsicmanner. The sequence of a cDNA encoding human MCP has been reported byLublin et al, J. Exp. Med., (1988) 168:181-194. The nucleotide sequenceof a cDNA encoding MCP is shown in FIG. 4B (SEQ ID NO: 6).

TABLE 2 Amino Acid Domain  1-34 Leader peptide 35-95 CCP  96-158 CCP159-224 CCP 225-285 CCP 286-314 STP B-domain: VSTSSTTKPASSAS C-domain:GPRPTYKPPVSNP 315-327 Undefined segment 328-351 Transmembrane domain352-361 Intracytoplasmic anchor 362-377 Cytoplasmic tail one:TYLTDETHREVKFTSL 362-384 Cytoplasmic tail two: KADGGAEYATYQTKSTTPAEQRC

Effects of Excessive Activation of Complement

Excessive activation of complement causes damage to normal host tissuesin a number of conditions. Some diseases in which complement is known tobe activated include systemic lupus erythematosus, acute myocardialinfarction, burn, sepsis, stroke and the adult respiratory distresssyndrome. Accordingly, it is desirable to have soluble agents that canblock complement activation. Such agents would be useful for treatingthe above-mentioned human diseases and a wide range of other diseases(See Table 3 below). The construction of hybrid complement regulatoryproteins has been attempted previously, but with mixed results. Forexample, a hybrid containing CCPs 1-4 of MCP and CCPs 1-4 of DAF wasconstructed by Iwata, et al (J. Immunol. 1194, 152:3436). While thishybrid had greater activity in the alternative pathway than either MCPor DAF, it had less activity than DAF alone or DAF plus MCP in theclassical pathway. Additionally, in tests of reciprocal chimericcomplement inhibitors, one chimeric protein retained the activity of tsCD59 and DAF components, while its reciprocal retained only the activityof its DAF component (Fodor, et al, J. Immunol., 1995, 155:4135).Therefore, there is a need for a reliable method for constructing hybridand chimeric complement regulatory proteins. There is also a need for ahybrid complement regulating protein that possesses the decayaccelerating activity of DAF and the co factor activity of CR1.

TABLE 3 Potential Clinical Targets of Protein of the InventionAlternative Pathway Classical Pathway Reperfusion injury Autoimmunediseases Cerebral infarction (stroke) Systemic lupus erythematosus Acutemyocardial infarction Rheumatoid arthritis Hypovolemic shockGlomerulonephritis Multiple organ failure Hemolytic anemia Crush injuryMyasthenia gravis Intestinal ischemia Multiple sclerosis Inflammatorydisorders Vasculitis Adult respiratory distress syndrome Inflammatorybowel diseases Thermal injury (burn & frostbite) Bullous diseasesPost-pump syndrome (cardiopulmonary Urticaria bypass & hemodialysis)Paroxysmal nocturnal Crohn's disease Hemoglobinuria Sickle cell anemiaCryoglobulinemia Pancreatitis Inflammatory disorders Adverse drugreactions Septic shock & endotoxemia Radiographic contrast media allergyTransplant rejection Drug allergy Hyperacute allograft IL-2 inducedvascular leakage syndrome Transplant rejection Hyperacute xenograft

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a listing of the amino acid sequence of human DecayAccelerating Factor (DAF), SEQ. ID NO: 1;

FIG. 1B is a listing of a DNA sequence encoding DAF, SEQ. ID NO: 2;

FIG. 2 is a listing of the amino acid sequence of human ComplementReceptor 1 (CR1), SEQ. ID NO: 3;

FIG. 3 is a listing of a DNA sequence encoding CR1, SEQ. ID NO: 4;

FIG. 4A is listing of the amino acid sequence of human Membrane CofactorProtein (MCP), SEQ. ID NO: 5;

FIG. 4B is a listing of a DNA sequence encoding MCP, SEQ. ID NO: 6;

FIG. 5 is a representation of a lipid tail structure;

FIGS. 6A and 6B are listings of PCR primers DSIGEB and DAF3P, SEQ. IDNO's: 7 and 8, respectively;

FIGS. 7A-7D are listings of PCR primers CR1094X, CR1099N, CR1350N, andCR1B3P, SEQ. ID NO's: 9-12, respectively;

FIG. 8A is a listing of the amino acid sequence of the protein DAF-CR1B,SEQ. ID NO: 13;

FIG. 8B is a listing of a DNA sequence encoding DAF-CR1B, SEQ. ID NO:14;

FIG. 9A is a listing of the amino acid sequence of protein DAF-CR1BB,SEQ. ID NO: 15;

FIG. 9B is a listing of a DNA sequence encoding DAF-CR1BB, SEQ. ID NO:15;

FIGS. 10A and 10B are listings of the PCR primers IgG45 and IgG43, SEQ.ID NO's: 17 and 18, respectively;

FIG. 11A is a listing of amino acid sequence of protein DAF-IgG4, SEQ.ID NO: 19;

FIG. 11B is a listing of a DNA sequence encoding DAF-IgG4, SEQ. ID NO:19;

FIGS. 12A and 12B are listings of the PCR primers MCP5 and MCP3, SEQ. IDNO's: 21 and 22, respectively;

FIG. 13A is a listing of the amino acid sequence of protein DAF-MCP, SEQID NO: 23;

FIG. 13B is a listing of a DNA sequence encoding DAF-MCP, SEQ. ID NO:24;

FIG. 14 is a Western blot of protein samples containing hybrid proteinsprobed with monoclonal antibodies raised against DAF and CR1;

FIG. 15 is a Western blot of a protein samples containing DAF-MCP probedwith monoclonal antibodies raised against DAF and MCP;

FIG. 16 is a graph showing the percent inhibition of hemolysis ofDAF-CR1BB and sCR1 in a whole serum assay;

FIG. 17 is a graph showing the percent inhibition of hemolysis ofDAF-MCP and DAF in a whole serum assay;

FIGS. 18A and 18B are graphs showing the percent inhibition of hemolysisof the hybrid proteins in a classical pathway C3 convertase assay;

FIG. 19 is a graph showing the percent inhibition of hemolysis ofDAF-CR1B and DAF in a classical pathway C5 convertase assay;

FIG. 20 is a graph showing the percent inhibition of hemolysis ofDAF-CR1BB, sCR1 and DAF-CR1B in a classical pathway C5 convertase assay;

FIG. 21 is a Western blot of supernatants of cells expressing the hybridproteins of DAF-MCP or DAF-CR1BB with and without factor I in a cofactorassay.

FIG. 22 is a Western blot of supernatants of cells expressing the hybridproteins of DAF-Cr1B with and without factor I in a cofactor assay.

SUMMARY OF THE INVENTION

It is therefore an aspect of the present invention to provide hybrid andchimeric complement regulating proteins. The present invention relatesto a family of hybrid and chimeric polypeptides for regulating, moreparticularly for inhibiting, excessive complement activation.

A hybrid complement-regulating protein of the present inventioncomprises a first functional unit of a first complement regulatoryprotein having complement regulating properties, a first spacer sequenceof at least about 200 amino acids encoding a polypeptide that does notexhibit complement regulating properties and at least a secondfunctional unit attached to the spacer sequence. The second functionalunit may be a polypeptide providing a functional unit of a secondcomplement regulatory protein, a polypeptide derived from animmunoglobulin, or a polypeptide that enhances binding of the protein toan animal cell. The hybrid protein may also contain a second spacersequence and a third functional unit of a complement regulatory protein,a polypeptide derived from an immunoglobulin, and a polypeptide thatenhances binding of the protein to an animal cell. The optional thirdfunctional unit may be the same or different from the first or secondfunctional units. It has been advantageously discovered that theconstruction of hybrid complement regulating proteins requires more thansimply the presence of the protein domains providing decay acceleratingactivity (as from DAF) and co-factor activity (as from CR1 or MCP).Proper spacing of such domains is also required for activity of bothdomains in a hybrid protein. In one embodiment, the hybrid protein(referred to hereafter as a “DAF hybrid”) comprises CCPs 2 and 3, of DAFas one functional unit. Preferably, such a functional unit alsocomprises CCP4 and more preferably, comprises CCPs 1-4 of DAF. The DAFhybrid protein can also include one or more functional units that havebeen derived from CR1, e.g., one or more functional units comprisingCCPs 8-10 of CR1 or functional units comprising CCPs 15-17 of CR1, orcombinations thereof. The DAF hybrid polypeptide can also include one ormore functional units that have been derived from MCP, e.g. CCPs 2, 3,and 4. Preferably, the MCP functional unit also comprises CCP1 of MCP.The DAF hybrid protein can also comprise functional units that have beenderived from other complement activation regulatory proteins. Examplesof such proteins include, but are not limited to, the factor H proteinand C4BP. In certain embodiments, the hybrid polypeptide comprisesfunctional units that have been derived from three or more complementactivation regulatory proteins, in which each functional unit isseparated from the preceding functional unit and following functionalunit in the hybrid polypeptide by a spacer.

The present spacer is a polypeptide that is greater than 200 amino acidsin length, preferably greater than 250 amino acids in length. Where morethan one spacer is used, the amino acid sequences of the spacers thatare employed may be the same or different. The spacer may be a syntheticpolypeptide fragment. Alternatively, the spacer is derived from acomplement activation regulatory protein. In one embodiment, the spacercomprises all or substantially all of CCPs 4-7 of CR1, i.e, amino acid239 through amino acid 496 of the CR1 sequence shown in FIG. 2 (SEQ. IDNO: 2). These CCPs have no known activity directly associated with them.While not wishing to condition patentability on any particular theory,it is believed that these CCPs function in the native CR1 protein toproperly space those CCPs that do have directly-associated activity fromeach other. In another embodiment, the spacer comprises all orsubstantially all of CCPs 11-14 of the CR1 protein. In most embodiments,the spacer does not exhibit complement-regulating activity. The hybridproteins of the present invention are based, at least in part, onApplicants discovery that on an equal molar basis, DAF is at least 4 to5 times more efficient than LHR A of CR1 in inhibiting the classicalpathway.

The chimeric polypeptides of the present invention comprise at least onefunctional unit that has been derived from a complement activationregulatory protein (referred to hereinafter as the “first functionalunit”), a functional unit that has been derived from a protein that isnot a complement activation regulatory protein (referred to hereinafteras the “second functional unit”), and a spacer for separating andappropriately spacing the first functional unit from the secondfunctional unit. The spacer is as described above. The second functionalunit can be derived from immunoglobulin (IgG) and may serve to reducedegradation of the chimeric polypeptide following injection into ananimal. Alternatively, the second functional unit can be a targetingmoiety that enhances binding of the chimeric polypeptide by certainanimal tissues. An example of one such targeting moiety is a lipid tail,as shown in attached FIG. 5. Such a molecule is expected to target thechimeric polypeptide to the membrane bilayer interior, more particularlyto areas of translocated acidic phospholipid. (See, Smith, R A (2002)Biochem Soc Trans 30 (Pt 6):1037-41.) The second function unit can alsobe a targeting moiety that enhances binding of the chimeric polypeptideto an implant, or to an extracorporeal surface, e.g., a hemodialysismembrane. In certain embodiments, the chimeric protein may comprisemultiple functional units that have been derived from one or morecomplement activation regulatory proteins, each of which are separatedfrom one another by a spacer. Thus, the chimeric polypeptide of thepresent invention can be a hybrid-chimeric polypeptide, e.g. apolypeptide that comprises a functional unit derived from DAF, afunctional unit that has been derived from CR1 and a functional unitthat has been derived from IgG 4.

The present invention also provides isolated polynucleotides that encodethe hybrid and chimeric polypeptides of the present invention,constructs formed by inserting an isolated polynucleotide of the presentinvention into an expression vector, and recombinant host cells intowhich the constructs of the present invention have been incorporated. Inaddition to the hybrid and/or chimeric polypeptide encoding sequence,such expression vectors comprise regulatory sequences that control orregulate expression of the polypeptide. Examples of suitable host cellsare bacterial cells, yeast cells, insect cells, and mammalian cells. Thepresent invention also relates to a process for preparing the hybridand/or chimeric proteins of the present invention by culturing the cellsof the present invention under conditions that promote expression of thehybrid and/or chimeric protein in the cell. For example, the process maybe carried out by expressing the hybrid or chimeric protein in Chinesehamster ovary (CHO) cells or COS cells. The hybrid and chimeric proteinsof the present invention may then be collected from a cell culturesupernatant or cell lysate of the transformed host cells using anaffinity column and then eluting the hybrid and/or chimeric protein fromthe column.

The present invention also features methods of reducing inflammationcharacterized by excessive complement activation in an animal subject.In one aspect, the method comprises administering one or more of thepresent hybrid polypeptides or chimeric polypeptides to an animalsubject, particularly a human subject, afflicted with a conditionassociated with excessive complement activation. Thus, the presentinvention also relates to methods of treating patients afflicted withany of the diseases listed in Table 3 below. In another aspect, thepresent method comprises administering an expression vector comprising apolynucleotide that encodes a hybrid polypeptide or a chimericpolypeptide of the present invention to the animal subject.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are hybrid proteins that comprise at least onefunctional unit of a first complement activation regulatory protein andat least one functional unit of a second complement activationregulatory protein, particularly a protein that inhibits the activity ofC3 and/or C5 convertase. In certain embodiments, such hybrid proteinscomprise more than one functional unit from a complement activationregulatory protein. In certain embodiments such hybrid proteinscomprises functional units from more than 2 complement activationregulatory proteins. The functional units in the present hybrid proteinsare separated and spaced apart by a spacer which is described in greaterdetail below. The functional units can be located in any order withinthe hybrid proteins of the present invention, provided that properspacing exists between the functional units.

I. Hybrid Proteins

A. DAF Functional Unit

In certain embodiments, the present hybrid protein preferably comprisesat least one functional unit from DAF. Such a functional unit is capableof dissociating C3 and C5 convertases. Thus, the DAF functional unit maycomprise CCPs 2 and 3 of DAF, which are sufficient for decayaccelerating activity against the classical pathway. Preferably, the DAFfunctional unit comprise CCPs 2, 3, and 4 of DAF, which are sufficientfor decay accelerating activity against both the classical pathway C3convertase and the alternative pathway C3 convertase. The amino acidsequence of such CCPs may be identical to the native or naturallyoccurring amino acid sequence of DAF. Alternatively, the amino acidsequence of such CCPs may be altered slightly, particularly at the aminoor carboxy terminus. Such alterations occur when a restriction enzymesite is incorporated into the polynucleotide encoding the CCPs. Suchalterations also occur when amino acids are deleted from the N terminusor C terminus of the functional unit; (For example, see Example 1 belowin which a number of amino acids are deleted from the C terminus of CCP4 of DAF.) In certain embodiments the hybrid protein may furthercomprise CCP 1 of DAF. It is also envisioned that some amino acidsubstitutions in the sequence may be introduced without effecting theactivity of the functional until as disclosed in U.S. Pat. No.6,521,450, the disclosure of which is hereby incorporated by referenceherein. Therefore, the functional until may have less than completehomolgy to native protein component. Changes may be made by substitutionof like charged amino acids for one another or substitution ofhydrophilic amino acids for one another substitution of hydrophobicamino acids for one another and substitution of amino acids of similarmass for one another. In other regions, especially, those unassociatedwith activity less subtly changes may be made. In one embodiment, theprotein contains a functional unit that has at least 95 percent homologyto a region of CCPs 1, 2, 3 or 4 of the human DAF protein. The aminoacid sequence of native DAF is shown in FIG. 1A (SEQ. ID NO: 1). CCP1extends from and includes amino acid 35 through amino acid 95 of thenative DAF protein. CCP2 extends from and includes amino acid throughamino acid 97 through amino acid 159 of the native DAF protein. CCP3extends from and includes amino acid 162-221 of the native DAF protein.CCP 4 extends from and includes 224-284 of the native DAF protein. Thefunctional unit also comprises the amino acids that link CCP1 to CCP2,CCP2 to CCP3, and CCP3 to CCP 4 of the DAF protein.

B. CR1 Functional Units

The present hybrid protein may also comprise one or more functionalunits from CR1. Such functional unit is capable of acting as a cofactorfor factor I-mediated cleavage of C3b to iC3b and C3f and furthercleavage of iC3b to C3c and C3 dg. In this way, a hybrid protein may notonly have strong activity in disssociating C3 convertase, but alsostrongly mediate further cleavage of the resulting 03b protein by factorI. Such a functional unit is also capable of acting as a cofactor forfactor I-mediated cleavage of C4b to C4d and Cr4c. (See K. Yazdanbakhshet al., Blood, 2003). Thus, the hybrid protein of the present inventionmay comprise substantially all of CCPs 8-10 and/or CCPs 15-17 of CR1.The amino acid sequence of such CCPs may be identical to the native ornaturally occurring amino acid sequence of CCPs 8-10 or CCPs 15-17 ofCR1. Alternatively, the amino acid sequence of such CCPs may be alteredslightly, particularly at the amino or carboxy terminus. Suchalterations occur when a restriction enzyme site is incorporated intothe polynucleotide encoding the CCPs 8-10 or CCPs 15-17 of CR1 or whenamino acids, preferably a few amino acids, such as 1 to 10 aa's aredeleted from the amino terminus or the carboxy terminus or both theamino terminus and the carboxy terminus of the functional unit. Aminoacid substitutions, as described above, may also be introduced into theCCPs of CR1. The hybrid protein of the present invention may comprisetwo or more functional units from CR1. Such CR1 functional units may bethe same or different. Thus, the hybrid protein of the present inventionmay comprise two functional units derived from LHR-B or one functionalunit derived from LHR B and one functional unit derived from LHR-C ofthe CR1 protein. CCP 8 of CR1 extends from and includes amino acid497-556 of the native CR1 protein. CCP 9 of CR1 extends from andincludes amino acids 557-618 of the native CR1 protein. CCP 10 extendsfrom and includes amino acids 619-688 of the native CR1 protein. CCP 15of CR1 extends from and includes amino acid 947-1006 of the native CR1protein. CCP 16 of CR1 extends from and includes amino acid 1007-1068 ofthe native CR1 protein. CCP 17 of CR1 extends from and includes aminoacid 1069-1138 of the native CR1 protein.

C. MCP Functional Unit

The present hybrid protein may also comprise one or more functionalunits from MCP. Such functional unit is capable of acting as a cofactorfor factor I-mediated cleavage of C3b to iC3b and C3f. Thus, the hybridprotein of the present invention may comprise substantially all of CCPs1-4 of MCP. The amino acid sequence of such CCPs may be identical to thenative or naturally-occurring amino acid sequence of CCPs 1-4 of MCP.Alternatively, the amino acid sequence of such CCPs may be alteredslightly, particularly at the amino or carboxy terminus, or withsubstitutions as described above. Such alterations occur when arestriction enzyme site is incorporated into the polynucleotide encodingCCPs 1-4 of MCP, or when amino acids are deleted from the amino orcarboxy terminus of this functional unit. Preferably, the hybrid proteinof the present invention comprises two or more functional units from MCPor units having at least 95 percent homology to two or more functionalunits from MCP, or alternatively, a functional unit from MCP and afunctional unit from CR1. CCP 1 of MCP extends from and includes aminoacid 35-95 of the native MCP protein. CCP 2 of MCP extends from andincludes amino acid 96-158 of the native MCP protein. CCP3 extends fromand includes amino acid 159-224 of the native MCP protein. CCP4 extendsfrom and includes amino acid 225-285 of the native MCP protein.

D. Spacer

The hybrid proteins of the present invention comprise one or morespacers. Each spacer in the present hybrid and/or chimeric proteinsseparate and appropriately space the functional units of the presenthybrid and/or chimeric proteins from one another. Such spacer is apolypeptide that is greater than 200 amino acids in length, preferablygreater than 250 amino acids in length. Where more than one spacer isrequired, the amino acid sequences of the spacers that are employed inthe hybrid proteins of the present invention may be the same ordifferent. In one preferred embodiment, the spacer comprises all orsubstantially all of CCPs 4-7 of CR1, i.e., amino acid 239 through aminoacid 496 of the CR1 sequence shown in FIG. 2 (SEQ. ID NO: 3). In anotherpreferred embodiment the spacer comprises all or substantially all ofCCPs 11-14 of the CR1 protein. As used herein the term substantially allmeans that the spacer may lack a few, e.g. 1-10 amino acids from the Nterminus and/or the C terminus of the spacer. The spacer may alsocomprise some amino acids that result from incorporating a restrictionenzyme site into the spacer. Thus, the spacer may comprise a few aminoacids at the N terminus or C terminus that are different from the aminoacids that are found at the N terminus or C terminus of the CCP4-7fragment that is derived from native CR1 or the CCP11-14 fragment thatis derived from native CR1. The spaced may also contain substitutionswithin a sequence as described above, such that the spacer has at leastabout 95 percent homology to a corresponding native sequence.

Optionally, the hybrid proteins of the present invention may furtherinclude a tag, i.e., a second protein or one or more amino acids,preferably from about 2 to 65 amino acids, that are added to the aminoor carboxy terminus of the hybrid protein. Typically, such additions aremade to stabilize the protein or to simplify purification of anexpressed recombinant form of the hybrid protein Such tags are known inthe art. Representative examples of such tags include sequences whichencode a series of histidine residues, the epitope tag FLAG, the Herpessimplex glycoprotein D, beta-galactosidase, maltose binding protein, orglutathione S-transferase.

The present invention also encompasses hybrid protein proteins in whichone or more amino acids are altered by post-translation processes orsynthetic methods. Examples of such modifications include, but are notlimited to, glycosylation, iodination, myristoylation, and pegylation.

II. Chimeric Proteins.

The chimeric proteins of the present invention comprise one or morefunctional units of complement activation regulatory protein asdescribed above and one or more functional units derived from a proteinthat is not a complement activation regulatory protein. Examples offunctional units that are not derived from complement activationregulatory proteins include functional units that are derived from animmunoglobulin, particularly IgG4, and that serve to reduce degradationof the chimeric polypeptide following injection into an animal. Thus,the chimeric protein may include the hinge, CH2, and CH3 domains ofIgG4. Alternatively, the second functional unit can be a targetingmoiety that enhances binding of the chimeric polypeptide by certainanimal tissues. An example of such targeting moiety is a lipid tail, asshown in attached FIG. 5. In certain embodiments, the chimeric proteinmay comprise multiple functional units that have been derived from oneor more complement activation regulatory proteins, each of which areseparated from one another by a spacer. Thus, the chimeric polypeptidesof the present invention can be a hybrid, chimeric polypeptide.

Preparation of the Hybrid and Chimeric Proteins

The present hybrid proteins and chimeric proteins of the presentinvention are prepared using polynucleotides that encode such proteinsand expression systems.

The functional units and spacers employed in the present hybrid and/orchimeric proteins can be made by obtaining total (t) or messenger (m)RNA from an appropriate tissue, cell line or white blood cells. SuitableRNA (total or messenger) is also available commercially. Blood can bedrawn from a human or other animal subject and peripheral bloodmononuclear cells (PBMCs) can be purified by Ficoll-Paque densitycentrifugation. Total RNA from PBMCs should contain CR1 and IgG4. Celllines can be grown in controlled climate incubators with appropriatecell culture media. DAF and MCP are fairly ubiquitous proteins. Thus,these proteins can be found in most cell lines, e.g., the HeLa cellline.

Following isolation of suitable RNA, the RNA is reverse transcribed tocDNA using commercially available reagents and standard protocols, e.g.,the Superscript protocol of Invitrogen. Once the appropriate cDNA ismade, polymerase chain reaction (PCR) can be used in conjunction withDNA polymerases and oligonucleotide primer pairs (20 to 30 nucleotidesin length) to amplify DAF, MCP, CR1 and/or IgG4 cDNA. One primer will beat the 5′ end of the cDNA (for example at the start codon ATG or, in thecase of the constant heavy chain, further downstream at the start of theconstant heavy region 1 [CH1] and one primer will be at the 3′ end ofthe cDNA, e.g. at the stop codon TAG, TAA, or TGA). The PCR products arethen subcloned into vectors such as pT7Blue (pT7B) (Novagen, Madison,Wis.) and sequenced to confirm that the correct cDNA was amplified.

Expression Systems for Producing the Hybrid Proteins

The present hybrid proteins can be produced in procaryotic andeucaryotic cells each using different expression vectors that areappropriate for each host cell. Eucaryotic expression system such as thebaculoviral or mammalian cells are described below.

The following are examples of expression vectors which may be used forgene expression in an eucaryotic expression system. The plasmid, pMSG,uses the promoter from mouse mammary tumor virus long terminal repeat(MMTV). Suitable host cells for pMSG expression are chinese hamsterovary (CHO) cells, HeLa cells and mouse Lkt negative cells (Lee, F., etal., 1981, Nature 294:228-232). The vector, pSVL, uses the SV40 latepromoter. For high transient expression, suitable host cells for pSVLexpression are COS cells (Sprague, J. et al., 1983, J. Virol.45:773-781). The vector, pRSV, uses Rous Sarcoma Virus promoter.Suitable host cells for pRSV expression are mouse fibroblast cells,lymphoblastoid cells and COS cells (Gorman, Padmanabhan and Howard,1983, Science 221:551-553).

Baculovirus expression vectors can also be used. These vectors arestably expressed in insect cells such as sf9 (Luckow, V. A. and Summers,M. D., 1988, Bio/Technology 6:47-55; Miller, L. K., 1988, Ann. Re.Microbiology 42:177-199).

Hybrid proteins of the invention can also be produced in a procaryoticexpression system. The following are examples of expression vectorswhich can be expressed in procaryotic expression systems. The pOXexpression series using the oxygen-dependent promoter can be expressedin E. coli. (Khosla, G., et al., 1990, Bio/Technology 8:554-558). pRLvector which uses the strong pL promoter of lambda phage (Reed, R. R.,1981, Cell 25:713-719; Mott, J. D., et al., 1985, Proc. Natl. Acad. Sci.U.S.A. 82:88-92) and the pKK223-3 vector which uses a hybrid promoterderived from the fusion between the promoters of the tryptophan andlactose operons of E. coli. (Borsius, J. and Holy, A., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6929-6933) can be used for expression in E. coli.

Suitable vectors for yeast expression are also well known in the art,e.g., Sleep, D., Belfield, D. P. and Goodey, A. R., 1990, Bio/Technology8:42-46; Sakai, A. et al., 1991, Bio/Technology 9:1382-1385; Kotula, L.and Curtis, P. J., 1991, Bio/Technology 9:1386-1389, all of which areherein incorporated by reference.

Production, Quantitation, Purification and Analysis of the HybridProteins.

Once a recombinant cell line that expresses the hybrid protein has beenisolated, the secreted proteins are identified and verified with regardto their predicted structure. Various methods can be used to identifyand characterize the expressed hybrid proteins. The presence of secretedhybrid proteins can be verified by immunoprecipitation with monoclonalantibodies to one or the other fragment, e.g., antibodies that bind toCCP's, 2, 3, or 4 of DAF or to LHR B or C of CR1.

Another method that could be used with present hybrid and/or chimericproteins is a double immunoprecipitation, using two monoclonalantibodies of different specificities in succession. Pre-clearance ofculture supernatant with one antibody would result in a negativeimmunoprecipitation with the second antibody. This method would verifythat a single protein expresses both CR1 and DAF epitopes.

Alternatively, the hybrid DAF-CR1 protein, can be identified by Westernblot. For example, after SDS-PAGE and transfer to nitrocellulose, blotscan be developed with either anti-CR1 antibodies or anti-DAF monoclonalantibodies. The expressed bispecific recombinant protein would bereactive with both antibodies, again demonstrating the presence ofspecific DAF and CR1 epitopes in the hybrid protein.

Identification of the present hybrid and/or chimeric proteins can alsobe accomplished by ELISA. For example, a rabbit polyclonal antibodyspecific for either LHR B or C of CR1 or CCP's 2, 3, or 4 of DAF can beused to coat plastic microtiter ELISA plates, followed by the additionof culture supernatant from the recombinant cell line expressing theDAF-CR1 hybrid and incubation with the capture polyclonal antibody. Amonoclonal anti-DAF or anti-CR1 second antibody, the specificity ofwhich is different from the capture antibody, can be subsequently used.A positive reaction would indicate the presence of both epitopes on thehybrid or chimeric protein.

An ELISA can also be used to quantitate the levels of the DAF-CR1 hybridprotein in culture supernatants or any other unpurified solutionscontaining the chimeric protein by comparison to standard curve of knownquantities of purified DAF-CR1 hybrid protein. Quantitation of DAF-CR1hybrid protein would be useful for determination of production rates inrecombinant cell lines, determination of protein concentration inpartially purified preparations, and for determination of proteinconcentration in plasma for in vivo experiments.

The hybrid and/or chimeric proteins of the present invention can bepurified from recombinant cell culture supernatant by a variety ofstandard chromatographic procedures, including but not limited toimmunoaffinity chromatography, ion exchange chromatography, gelfiltration chromatography, reverse-phase high pressure liquidchromatography (HPLC), lectin affinity chromatography, orchromatofocusing. For example, small quantities of culture supernatantcontaining serum supplement can be purified using immunoaffinitychromatography with, e.g., anti-CR1 or anti-DAF monoclonal antibodies.DAF-CR1 hybrid protein bound to the immobilized antibody can be elutedin purified form by use of a chaotropic solution.

Once the hybrid and/or chimeric protein is purified, its amino acidsequence can be deduced by direct protein sequence analysis using anautomated system. The presence of N— and O-linked carbohydrates can bedetermined by use of specific endoglycosidase enzymes (Chavira, R. etal., 1984, Anal. Biochem. 136:446). Further characterizations of itsbiochemical structure can also be performed, including but not limitedto pI determination by isoelectric focusing, hydrophilicity analysis,X-ray crystallographic analysis, and computer modeling.

Functional Characterization of the Present Hybrid and/or ChimericProteins

The hybrid proteins of the present invention have the ability tofunction both as a cofactor for Factor I and as a decay acceleratingfactor. In vitro assays can be performed to measure these biologicalactivities (Medof, M. et al., 1984, J. Exp. Med. 160:1558; Masaki, T. etal., 1992, J. Biochem 111:573). As described in the examples, assays forcofactor activity and for decay accelerating activity are used todemonstrate both these complement regulatory functions for the presenthybrid protein. The consequence of either cofactor or decay acceleratingactivity, or in the case of a DAF-CR1 or DAF-MCP hybrid protein, bothactivities in combination, is the inactivation of C3/C5 convertases.Another suitable in vitro assay demonstrates that the present hybridprotein is capable of inhibiting C5 convertase activity as measured bythe production of C5a Moran, P. et al., 1992, J. Immunol. 149:1736,herein incorporated by reference). Additional assays, as described inthe examples below, demonstrate that the present hybrid proteins inhibitthe complement-induced lysis of cells via the classical and alternativepathways.

Demonstration of In Vivo Therapeutic Activity of the Present Hybridand/or Chimeric Proteins

The Arthus reaction is an inflammatory response caused by theinteraction of antigen in tissue with circulating antibody. It has beenused as a classic example of a localized in vivo inflammatory response,and is characterized by the formation of immune complexes, complementactivation, inflammatory cell recruitment, edema and tissue damage(Bailey, P. and Sturm, A., 1983, Biochem. Pharm 32:475). Experimentally,a reversed passive Arthus reaction can be established in an animal modelby i.v. injection with antigen and subsequent challenge with antibody.Using guinea pigs as an animal model, the in vivo therapeutic efficacyof the hybrid and/or chimeric proteins of the invention can beevaluated.

Additional animal models with relevance to various clinical humandiseases can also be used to test the in vivo efficacy of complementactivation blockers. These include, but are not limited to: myocardialischemia/reperfusion injury (acute myocardial infarction; Weisman, H. F.et al., 1990, Science 249:146); cerebral ischemic injury (stroke; Chang,L. et al., 1992, J. Cerebr. Blood Flow Metab. 12:1030); lung injury(ARDS; Hosea, S. et al., 1980, J. Clin. Invest. 66:375); xenograftrejection (transplants; Leventhal, J. et al., 1993, Transplantation55:857); burn injury (Caldwell, F, et al., 1993, J. Burn Care Rehab.14:420); acute pancreatitis (Steer, M. 1992, Yale J. Biol. Med. 65:421),nephritis (Pichler, R. et al., 1994, Am. J. Pathol. 144:915),cardiopulmonary bypass (Nilsson, L. et al., 1990, Artif. Organs 14:46),and multiple sclerosis (Linington, C. et al., 1989, Brain 112:895).

Administration of the Present Hybrid and Chimeric Proteins to AnimalSubjects

The present hybrid and chimeric proteins can be combined with anappropriate pharmaceutical formulation and administered to an animalsubject, particularly a human subject, by a variety of routes,including, but not limited to, intravenous bolus injection, intravenousinfusion, intraperitoneal, intradermal, intramuscular, subcutaneous, andintranasal routes. The administration of the present hybrid proteins invivo will enable the protein to bind endogenous C3/C5 convertases andinhibit the generation of additional C3b and C5b, of C3a and C5aanaphylatoxins, and of C5b-9 lytic complexes. The complement regulatoryactivities of the present hybrid proteins can therefore function toinhibit in vivo complement activation and the inflammatory sequelae thataccompany it, such as neutrophil recruitment and activation, autolysisof host cells, and edema. The present hybrid and/or chimeric proteinscan be used for the therapy of diseases or conditions that are mediatedby inordinate and/or excessive activation of the complement system.These include, but are not limited to: tissue damage due toischemia-reperfusion following myocardial infarction, aneurysm, stroke,hemorrhagic shock, or crush injury; burns; endotoxemia and septic shock;adult respiratory distress syndrome (ARDS); hyperacute rejection ofgrafts; cardiopulmonary bypass and pancreatitis. Autoimmune disordersincluding, but not limited to, systemic lupus erythematosis, rheumatoidarthritis, and multiple sclerosis, can also be treated with the hybridand/or chimeric proteins of the invention (also see Table 3).

Various delivery systems are known and can be used to deliver the hybridand/or chimeric proteins of the invention, such as encapsulation inliposomes, or controlled release devices. The hybrid and/or chimericproteins of the invention can also be administered extracorporeally,e.g., pre-conditioning donor organs prior to transplantation. The hybridand/or chimeric proteins of the invention can be formulated in apharmaceutical excipient in the range of approximately 10 μg/kg and 10mg/kg body weight for in vivo or ex vivo treatment.

Administration of Polynucleotides that Encode the Present Hybrid and/orChimeric Proteins to an Animal Subject

The present invention also relates to therapeutic methods in whichpolynucleotides that encode and express the present hybrid and/orchimeric polypeptides are introduced into a subject in need of the same,i.e. a subject, particularly a human subject, with a disorder associatedwith increased complement activation. Polynucleotides encoding andexpressing one or more hybrid and/or chimeric polypeptide can beintroduced into cells of the subject using any of a variety of methodsknown in the art to achieve transfer of DNA molecules into cells. Forexample, DNA encoding and expressing the hybrid and/or chimericpolypeptide can be incorporated into liposomes and targeted to andinternalized by the cells of the subject. Polynucleotides encoding thehybrid and/or chimeric polypeptide can also be incorporated intoplasmids that are introduced into cells of the subject by transfection.The hybrid and/or chimeric polypeptide encoding polynucleotides can alsobe introduced into cells using viruses. Such viral “vectors” can haveDNA or RNA genomes. Numerous such viral vectors are well known to thoseskilled in the art. Viral vectors that have polynucleotide sequencesencoding a DAF-CR1 hybrid polypeptide, for example, cloned into theirgenomes are referred to as “recombinant” viruses. Transfer of DNAmolecules using viruses is particularly useful for transferringpolynucleotide sequences into particular cells or tissues of an animal.Such techniques are commonly known in the art as gene therapy.

Expression vectors normally contain sequences that facilitate geneexpression. An expression vehicle can comprise a transcriptional unitcomprising an assembly of a protein encoding sequence and elements thatregulate transcription and translation. Transcriptional regulatoryelements generally include those elements that initiate transcription.Types of such elements include promoters and enhancers. Promoters may beconstitutive, inducible or tissue specific. Transcriptional regulatoryelements also include those that terminate transcription or provide thesignal for processing of the 3′ end of an RNA (signals forpolyadenylation). Translational regulatory sequences are normally partof the protein encoding sequences and include translational start codonsand translational termination codons. There may be additional sequencesthat are part of the protein encoding region, such as those sequencesthat direct a protein to the cellular membrane, a signal sequence forexample.

The hybrid and/or chimeric polypeptide encoding polynucleotides that areintroduced into cells are preferably expressed at a high level (i.e.,the introduced polynucleotide sequence produces a high quantity of thehybrid and/or chimeric polypeptide within the cells) after introductioninto the cells. Techniques for causing a high-level of expression ofpolynucleotide sequences introduced into cells are well known in theart. Such techniques frequently involve, but are not limited to,increasing the transcription of the polynucleotide sequence, once it hasbeen introduced into cells. Such techniques frequently involve the useof transcriptional promoters that cause transcription of the introducedpolynucleotide sequences to be initiated at a high rate. A variety ofsuch promoters exist and are well known in the art. Frequently, suchpromoters are derived from viruses. Such promoters can result inefficient transcription of polynucleotide sequences in a variety of celltypes. Such promoters can be constitutive (e.g., CMV enhancer/promoterfrom human cytomegalovirus) or inducible (e.g., MMTV enhancer/promoterfrom mouse mammary tumor virus). A variety of constitutive and induciblepromoters and enhancers are known in the art. Other promoters thatresult in transcription of polynucleotide sequences in specific celltypes, so-called “tissue-specific promoters,” can also be used. Avariety of promoters that are expressed in specific tissues exist andare known in the art. For example, promoters whose expression isspecific to neural, liver, epithelial and other cells exist and are wellknown in the art. Methods for making such DNA molecules (i.e.,recombinant DNA methods) are well known to those skilled in the art.

In the art, vectors refer to nucleic acid molecules capable of mediatingintroduction of another nucleic acid or polynucleotide sequence to whichit has been linked into a cell. One type of preferred vector is anepisome, i.e., a nucleic acid capable of extrachromosomal replication.Other types of vectors become part of the genome of the cell into whichthey are introduced. Vectors capable of directing the expression ofinserted DNA sequences are referred to as “expression vectors” and mayinclude plasmids, viruses, or other types of molecules known in the art.

Typically, vectors contain one or more restriction endonucleaserecognition sites which permit insertion of the hybrid polypeptideencoding sequence. The vector may further comprise a marker gene, suchas for example, a dominant antibiotic resistance gene, which encodecompounds that serve to identify and separate transformed cells fromnon-transformed cells.

One type of vector that can be used in the present invention is selectedfrom viral vectors. Viral vectors are recombinant viruses which aregenerally based on various viral families comprising poxviruses,herpesviruses, adenoviruses, parvoviruses and retroviruses. Suchrecombinant viruses generally comprise an exogenous polynucleotidesequence (herein, a polynucleotide encoding the hybrid and/or chimericpolypeptide) under control of a promoter which is able to causeexpression of the exogenous polynucleotide sequence in vector-infectedhost cells.

One type of viral vector is a defective adenovirus which has theexogenous polynucleotide sequence inserted into its genome. The term“defective adenovirus” refers to an adenovirus incapable of autonomouslyreplicating in the target cell. Generally, the genome of the defectiveadenovirus lacks the sequences necessary for the replication of thevirus in the infected cell. Such sequences are partially or, preferably,completely removed from the genome. To be able to infect target cells,the defective virus contains sufficient sequences from the originalgenome to permit encapsulation of the viral particles during in vitropreparation of the construct. Other sequences that the virus containsare any such sequences that are said to be genetically required “incis.”

Preferably, the adenovirus is of a serotype which is not pathogenic forman. Such serotypes include type 2 and 5 adenoviruses (Ad 2 or Ad 5). Inthe case of the Ad. 5 adenoviruses, the sequences necessary for thereplication are the E1A and E1B regions. Methods for preparingadenovirus vectors are described in U.S. Pat. No. 5,932,210, U.S. Pat.No. 5,985,846, and U.S. Pat. No. 6,033,908.

More preferably, the virus vector is an immunologically inertadenovirus. As used herein the term “immunologically inert” means theviral vector does not encode viral proteins that activate cellular andhumoral host immune responses. Methods for preparing immunologicallyinert adenoviruses are described in Parks et al., Proc Natl Acad Sci USA1996; 93(24) 13565-70; Leiber, A. et al., J. Virol. 1996; 70(12)8944-60; Hardy s., et al, J. Virol. 1997, 71(3): 1842-9; and Morsy etal, Proc. Natl. Acad. Sci. USA 1998. 95: 7866-71, all of which arespecifically incorporated herein by reference. Such methods involveCre-loxP recombination. In vitro, Cre-loxP recombination is particularlyadaptable to preparation of recombinant adenovirus and offers a methodfor removing unwanted viral nucleotide sequences. Replication deficientrecombinant adenovirus lacks the E1 coding sequences necessary for viralreplication. This function is provided by 293 cells, a human embryonickidney cell line transformed by adenovirus. First generationadenoviruses are generated by co-transfecting 293 cells with a helpervirus and a shuttle plasmid containing the foreign gene of interest.This results in the packaging of virus that replicates both the foreigngene and numerous viral proteins. More recently, 293 cells expressingCre recombinase, and helper virus containing essential viral sequencesand with a packaging signal flanked by loxP sites, have been developed(See Parks et al.) In this system, the helper virus supplies all of thenecessary signals for replication and packaging in trans, but is notpackaged due to excision of essential sequences flanked by loxP. When293-Cre cells are co-transfected with this helper virus, and a shuttleplasmid (pRP1001) containing the packaging signal, nonsense “fillerDNA”, and the foreign gene, only an adenovirus containing filler DNA andthe foreign gene is packaged (LoxAv). This results in a viralrecombinant that retains the ability to infect target cells andsynthesize the foreign gene, but does not produce viral proteins.

Another type of viral vector is a defective retrovirus which has theexogenous polynucleotide sequence inserted into its genome. Suchrecombinant retroviruses are well known in the art. Recombinantretroviruses for use in the present invention are preferably free ofcontaminating helper virus. Helper viruses are viruses that are notreplication defective and sometimes arise during the packaging of therecombinant retrovirus.

Non-defective or replication competent viral vectors can also be used.Such vectors retain sequences necessary for replication of the virus.Other types of vectors are plasmid vectors.

The methods also involve introduction of polynucleotides encoding thepresent hybrid and/or chimeric polypeptides into an animal subject inthe context of cells (e.g., ex vivo gene therapy).

EXAMPLES

The following examples contained herein are intended to illustrate butnot limit the invention.

Example 1 Hybrid Protein DAF-CR1B

A hybrid protein, DAF-CR1B, comprising a decay accelerating functionalunit derived from DAF, a cofactor 1 functional unit derived from CR1,and a spacer comprised of CCPs 4-7 of CR1 was made by recombinanttechniques. The DAF portion of the hybrid protein DAF-CR1B wasconstructed using DAF13.2.l/pBTKS and two primers DSIGEB and DAF3P in aPCR reaction (Vent polymerase [New England Biolabs] with the followingtimes: 94° C. 3 min [initial melting]; 94° C. 1 min, 55° C. 1 min 15sec, 72° C. 1 min 15 sec for 25 cycles; and 72° C. 7 min [finalextension]). DSIGEB is a 42 nucleotide (“nt”) primer that has thesequence 5′-ATA TAC GAA TTC AGA TCT ATG ACC GTC GCG CGG CCG AGC GTG-3′(FIG. 6A SEQ. ID NO:7). DAF3P is a 35nt primer that has the sequence5′-ACA GTG CTC GAG CAT TCA GGT GGT GGG CCA CTC CA-3′ (FIG. 6B, SEQ. IDNO:8). The resultant PCR product was named DAF1. It contained DAF'ssignal sequence followed by CCPs 1, 2, 3 and 4 ending with cysteine 249(Cys-249) in CCP4. Upstream of the signal sequence, two restrictionenzyme sites were built in, BglII.(A ▾ GATCT) and 5′ of BglII, EcoRI (G▾ AATTC). Three prime (3′) of CCP4 and encompassing part of the Cys-249codon (TGC), the restriction enzyme site XhoI (C ▾ TCGAG) was inserted.DAF1 was subcloned into pT7B and fully sequenced.

The CR1 portion of the hybrid protein DAF-CR1B was constructed usingCR1/AprM8. CR1/AprM8 was cut with the restriction enzyme NsiI (ATGCA ▾T) releasing several pieces, two of which were recovered (1094nts and1350nts) and subcloned into pGEM7Zf(+). The “1094” fragment(encompassing nts. 557 to 1670 of CR1) was amplified by PCR using theprimers CR1094X(5′) and CR1094N(3′). CR1094X is a 41nt primer having thesequence 5′-ATA TAC CTC GAG TCC TAA CAA ATG CAC GCC TCC AAA TGT GG-3′(FIG. 7A, SEQ ID NO:9). It has an XhoI site. CR1094N is a 34nt primerhaving the sequence 5′-ACA GTG ATG CAT TGG TTT GGG TTT TCA ACT TGG C-3′(FIG. 7B, SEQ ID NO:10). It has an NsiI site. This set of primersproduces a sequence from the linker between CCP3 and CCP4 of CR1 intoCCP8 of CR1. PCR conditions were the same as those for DAF1. The “1350”fragment, encompassing nts 1671 to 3020 of CR1, was amplified by PCRusing primers CR1350N(5′) and CR1B3P(3′). CR1350N is a 41nt primerhaving the sequence 5′-ATA TAC ATG CAT CTG ACT TTC CCA TTG GGA CAT CTTTAA AG-3′ (FIG. 7C, SEQ ID NO: 11). It has an NsiI site. CR1B3P is a57nt primer having the sequence 5′-ACA GTG AGA TCT TTA GTG ATG GTG ATGGTG ATG AAT TCC ACA GCG AGG GGC AGG GCT-3′ (FIG. 7D, SEQ ID NO: 12). Ithas a BglII site. PCR conditions were the same as those for DAF1 exceptthe 25 cycle extension time at 72° was 2 min, not 1 min 15 sec. This setof primers produces a sequence from CCP8 of CR1 to the end of CCP14 (inLHRB, specifically, . . . SSPAPRCGI) with a C-terminal 6×His tag andstop codon. These PCR fragments were subcloned into pT7B.

It is noteworthy that the natural linker between CCP3 and CCP4 of CR1 isthe amino acid sequence “IIPNK” (see FIG. 2, SEQ. ID NO: 3). Due to theinsertion of the XhoI restriction site, the hybrid protein's linkerbetween DAF CCP4 and CR1 CCP4 is “SSPNK” (see FIG. 8A (SEQ. ID NO: 13).

DNA sequence the data obtained confirmed the presence of the correctnucleotide sequences.

The vector pSG5 (Stratagene) was cut with the restriction enzymes EcoRIand BglII to accommodate the insertion of DAF1 (EcoRI to XhoI), CR828XN3(XhoI to NsiI) and CR1300NBF (NsiI to BglII). The vector and the threefragments were ligated using Promega T4 DNA ligase, and transformed intoDH5α maximum efficiency competent cells. Agarose gel electrophoresisconfirmed the presence of the vector and insert. The cDNA from onecolony was used for transfection into COS cells using Lipofectamine(Invitrogen) reagent. The supernatant was harvested two days later.Western blots using 2H6, an anti-DAF CCP4 antibody, and an anti-His tagantibody indicated the presence of the hybrid protein. The amino acidsequences of the DAF-CR1B is provided in FIG. 8A (SEQ. ID NO: 13). A DNAsequence encoding DAF-CR1B is provided in FIG. 8B (SEQ. ID NO: 14).

Example 2 Hybrid Protein DAF-CR1BB

Another hybrid protein, DAF-CR1BB, was prepared by recombinanttechniques. DAF-CR1BB, comprises DAF's four CCPs, a spacer comprised ofCCPs 4-7 of CR1, separating the functional unit of DAF from a firstcofactor 1 functional unit of CR1, LHR B, and a second spacer, CCPs11-14 of CR1, separating the first cofactor 1 functional unit of CR1from the second cofactor 1 functional unit of CR1, also LHR B. Morespecifically, DAF-CR1BB was prepared by adding an additional LHRB of CR1to DAF-CR1B To add the additional cofactor LH, DAF-CR1B was cut withBamHI and a BamHI fragment (nucleotide #1861 to 3210) from CR1 in AprM8was introduced. The BamHI fragment could enter the plasmid in either thecorrect or reverse orientation. Screening with SmaI found several cloneswith the correct nucleotide orientation. The amino acid sequence ofDAF-CR1BB is provided in FIG. 9A (SEQ ID NO: 15). The DNA sequenceencoding DAF-CR1BB is provided in FIG. 9B (SEQ ID NO: 16)

Example 3 Chimeric Protein DAF-IgG4

To increase half life of the hybrid protein, while minimizing complementactivation, as a starting point, part of the constant heavy region ofIgG4 was amplified by PCR and connected 3′ to nucleotides encoding adecay accelerating functional unit derived from DAF, and a spacerderived from CR1. The resulting protein DAF-IgG4, is composed of DAFCCPs1, 2, 3, 4+CR1 CCP4, 5, 6, 7 (part of LHRA)+IgG4, last amino acid(valine) of CH1-Hinge-CH2-CH3. The domains of IgG4 were amplified by PCRfrom pHC-huCg4, a gift of Gary McLean, 2222 Health Sciences Mall,Vancouver, B.C., Canada. The primers used in this PCR reaction were:

(FIG. 10A (SEQ ID NO: 17)) IgG45: 5′-ATA TAC GAA TTC TGG TTG AGT CCA AATATG GTC CC-3′ and (FIG. 10B (SEQ ID NO: 18)) IgG43: 5′-ACA GTG AGA TCTTTA TCA TTT ACC CGG AGA CAG GGA G-3′.

Per 100 μl PCR reaction, 2U Vent polymerase (New England Biolabs), 71.5ng pHC-Cg4 (7157 bp), 50 pmol of each primer, and 10 mM of each dNTPwere used. PCR settings were 1 initial denaturing cycle of 94° C. 3 min;25 cycles of 94° C. 1 min, 55° C. 1 min 15 sec, and 72° C. 1 min; and afinal extension cycle of 72° C. 7 min. A 700 bp fragment was recoveredwith the QIAquick gel purification kit. The fragment “IgG4 PCR” wasligated into the pT7Blue (pT7B) blunt vector (Novagen). Nova Blue(Novagen) and XL1Blue (Stratagene) competent cells were transformed withthe ligation mixture and plated on Ampicillin 50, Tetracycline 15,IPTG/Xgal LB agar plates. DNA sequence data confirmed the presence ofthe correct nucleotide sequence. A DNA sample was subsequently cut withEcoRI (“E”) and BglII (“B”) in Promega Buffer H (“H”) and the 700 bpband was purified with QIAquick. IgG41 E/B and pSG5 E/B/H were ligatedusing the Quick Ligation method (New England Biolabs) and transformedinto DH5αmax (Invitrogen) competent cells. The presence of the correctinserts was confirmed by digestion with EcoRI and BglII in Buffer H(Promega).

The resulting plasmid was cut with EcoRI (˜4800 bp linearized),purified, shrimp alkaline phosphatase (“SAP”)-treated, purified again,and quick ligated to an/EcoRI-cut fragment (gel purified, 1650 bp) whichcontains the DAF and CR1 portions of the sequence. DH5αmax competentcells were transformed with the ligation mix. Plasmid DNA from resultingcolonies were screened by cutting the DNA with BglII and examining theresulting band by agarose gel electrophoresis. Plasmid DNA was purifiedand cDNA was checked (uncut and BglII-cut). The amino acid sequence ofDAF-IgG4 is provided in FIG. 11A (SEQ. ID NO: 19). The DNA sequenceencoding DAF-IgG4 is provided in FIG. 11B (SEQ. ID NO: 20) The cDNA wastransfected into COS cells.

Note that the IgG45 primer codes for a slightly different link betweenCR1 CCP7 and the Hinge of IgG4 (in DAF-IgG4) than the link between CR1CCP7 and MCP CCP1 (in “DAF-MCP” see Example 4). IgG45 results in “GILV”(“V” is the last amino acid of the CH1 domain of IgG4) instead of“GILGH” which is found in DAF-MCP and is also the normal link betweenCR1 CCP7 and CCP8 (and is therefore what is found in DAF-CR1B andDAF-CR1BB hybrids).

Example 4 Hybrid Protein DAF-MCP

A hybrid protein, referred to hereafter as DAF-MCP, comprising a decayaccelerating functional unit of DAF, a cofactor 1 functional unitderived from MCP, and a spacer derived from CR1 was prepared (DAF CCPs1,2,3,4-CR1 CCPs 4,5,6,7-MCP CCPs 1,2,3,4+2 amino acids (VS) of MCP STPregion+6×His). MCP cDNA (with 3′-end sequence encoding GPI-anchoraddition) in PEE14 was used. More MCP cDNA in DH5α (Wizard SVDAF-IgG4-prep) (“MCP-GPI (A)”) was subsequently prepared. Primers forthe MCP portion of DAF-MCP are:

(FIG. 12A, SEQ ID NO: 21) MCP5: 5′-ATA TAC GAA TTC TGG GTC ACT GTG AGGAGC CAC CAA CAT TTG AAG C-3′; and (FIG. 12B, SEQ. ID NO: 22) MCP3:5′-ACA GTG AGA TCT TTA GTG ATG GTG ATG GTG ATG CGA CAC TTT AAG ACA CTTTGG AAC-3′.

The PCR reaction used Vent polymerase from New England Biolabs. The MCPPCR fragment was cut with EcoRI (“E”) and BglII (“B”) in Promega BufferH (“H”). The Quick Ligase method (New England Biolabs) was used toligate E/B/H-cut MCP PCR and E/B/H-cut pSG5. DH5α maximum efficiencycompetent cells were transformed with the mixture. Colonies were picked,the DNA was extracted from the bacteria and cut with E/B/H. All had aninsert. Two DNA samples were sequenced. An error at Nucleotide 680 wasfound which changes the amino acid at that position. Primers were madeto perform site-directed mutagenesis to correct the error (not shown).Subsequent DNA sequence data confirmed the correct sequence.

The resulting DNA was cut with EcoRI in Buffer H and purified withQiagen PCR purification kit. The purification product was treated withshrimp alkaline phosphatase (“SAP”), and purified again. Quick ligationmethod was used to ligate EcoRI- and SAP-treated MCP1A/pSG5 andEcoRI-treated and gel purified 1650 base pair piece from DAF-CR1BA whichadds the DAF and CR1 portions of the cDNA. After transformation, plasmidDNA was cut with BglII or with EcoRI to confim the presence and correctorientation of the insert. The amino acid sequence of DAF-MCP isprovided in FIG. 13A (SEQ. ID NO: 23). A DNA sequence of DAF-MCP isprovided in FIG. 13B (SEQ. ID NO: 24).

DNA was used for COS cell transfection. DAF-MCP 5A protein wasvisualized by western blot with monoclonal antibodies IA10 (against DAFCCP1) and GB24 (against MCP CCPs 3 and 4) (not shown). Size was between90 kDa and 100 kDa.

Testing of the Hybrid Proteins

Hemolytic Assays

Hemolytic assays are performed to assess the activity of the componentsof the classical and alternative pathways of complement or the activityof the regulators of complement activation (RCA) proteins. Classicalpathway activity is assessed using antibody-sensitized sheeperythrocytes (EshA) and can be undertaken in a variety of ways. Wholeserum can be used, or purified components of the complement cascade canbe used in a classical pathway C3 or C5 convertase hemolytic assay.

Experiments were undertaken using normal human, pig and rat serum toassess animal models for study. Human DAF has been found to be activeagainst convertases formed in pig serum (J. M. Perez de la Lastra et al.2000, J. Immunol. 165:2563). It is active at high concentrations againstconvertases formed in rat serum (C. L. Harris et al., 2000, Immunology100:462). In contrast, human CR1 is highly active against convertasesformed in rat serum. Classical pathway C3 convertase and C5 convertaseassays utilizing purified human complement components were also done tocompare the hybrids' performance to soluble DAF and soluble CR1.

Whole Serum Experiments

Serum (human, pig or rat) was titrated to a Z score of approximately 1,defined as an average of 1 lesion per sheep cell (A. P. Gee, 1983,“Molecular Titration of Components of the Classical Complement Pathway”in Methods in Enzymology 93:339). To each tube was added 1×10⁷ EshA, anRCA protein and the serum in DGVB⁺⁺ (dextrose gelatin veronal bufferwith calcium and magnesium) to a total volume of 200 ul. This mixturewas shaken for 30 min in a 37° C. water bath. GVBE (gelatin veronalbuffer with EDTA) was subsequently added to stop the hemolytic reaction.The extent of lysis was determined by reading the OD₄₁₂ of thehemoglobin red supernatant.

Classical Pathway C3 Convertase Hemolytic Assay

EshA (1×10⁷ cells/tube) were sequentially shaken and spun down with 30SFU (site-forming units) of human C1 (ART) for 15 min, 15SFU of human C4(Quidel) for 20 min, and sufficient human C2 (ART) for a Z score of 1for 5 min, all at 30° C. Following formation of the classical pathway C3convertase C4b2a, regulators were added for 15 min at 30° C. to assesstheir relative decay-accelerating activity. Guinea pig serum in GVBE(1:40 dilution) was subsequently added for 1 hr at 37° C. to allowformation of the terminal membrane complex. The cells were spun down andthe OD₄₁₂ of the hemoglobin red supernatant was read to determine theextent of cell lysis.

Classical Pathway C5 Convertase Hemolytic Assay

The classical pathway C5 convertase hemolytic assay was run over a 2-dayperiod. EshA (1×10⁷ cells/tube) were shaken sequentially with 60SFU ofhuman C1 (ART) for 15 min, 60SFU of human C4 (Quidel) for 20 min, and 10SPU human C2 (calculated after decay in a C3 convertase assay) (ART)along with 15SFU of human C3 (gift of C. Mold) for 5 min, all in a 30°C. water bath, to form the classical pathway C5 convertase C4b3b2a.Following this loading of complement components, 200 ul DGVB++ was addedto each tube and the tubes were shaken for 2 hr in a 30° C. water bathto decay the C2. The cells were spun down, resuspended in DGVB++, anddecay continued overnight with the cells at 4° C. The following day, thecells were shaken with 60SFU of human C1 for 15 min in a 30° C. waterbath, followed by sufficient human C2 for a Z score of 1 (5 min, 30°C.). RCA proteins were added for 15 min at 30° C. to accelerate thedecay of the C5 convertase. Subsequently, human C5 (Quidel) (1:250dilution) was added for 5 min at 30° C., then guinea pig C6-9 in DGVBE(dextrose gelatin veronal buffer with EDTA) (1:150 dilution) for 1 hr at37° C. Cells were spun down and the OD₄₁₂ of the hemoglobin redsupernatant was read to determine the extent of cell lysis.

Cofactor Experiments

The RCA proteins CR1, factor H and membrane cofactor protein MCP can actas cofactors for the factor I cleavage of C3b to smaller fragments(reviewed in M. Botto, “C3” [p. 88] in The Complement FactsBook, ed. B.J. Morley and M. J. Walport, 2000, San Diego: Academic Press). Thecofactor activity of CR1 resides in its LHRs B and C (S. C. Makrides etal., 1992, J. Biol. Chem. 267:24754; K. R. Kalli et al., 1991, J. Exp.Med. 174:1451; M. Krych et al., 1994, J. Biol. Chem. 269:13273). Allthree regulators can allow factor I to cleave C3b to iC3b. Factor I withCR1 can additionally cleave to iC3b to C3c. When the disulfide bridgesare reduced, an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) gel will show the following bands:

C3b: α′ (115 kDa), β (75 kDa)

iC3b: α′-68, α′-43, β (75 kDa), and a small soluble fragment C3f

C3c: α′-27, α′-40, β (75 kDa), and “membrane-attached” C3dg (40 kDa)

(A. Sahu et al., 1998, J. Immunol. 160:5596; A. M. Rosengard et al.,2002 PNAS 99:8808).

To confirm that the hybrid retained the cofactor activity of CR1, humanC3b (ART Lot 20P, 20 ng), was mixed with human factor I (ART Lot 6P, 60ng). Factor H (500 ng) or DAF-CR1BB (20 ng) or a control of 10 mM PO4buffer with 145 mM NaCl, pH 7.3 was added for a total volume of 10 uland incubated for 19 hr in a 37° water bath.

Samples of DAF-CR1B, DAF-CR1BB, and DAF-IgG4 separated on a 5% SDSpolyacrylamide gel and were examined by Western blot using monoclonalantibodies raised against DAF (IA10) or CR1 (E11) as shown in FIG. 14.Results indicated that the expressed hybrid proteins were recognized byantibodies to DAF or CR1 as expected.

A sample containing DAF-MCP was separated by 10% SDE PAGE and analyzedby Western blot using a monoclonal antibody raised against MCP (GB24) orDAF (IA 10), as shown in FIG. 15. The expressed DAF-MCP protein wasrecognized by GB 24 as expected.

Whole serum hemolytic assays were used to test the ability of the hybridproteins to inhibit complement activity. FIG. 16 shows the percentinhibition of hemolysis of DAF-CR1BB and SCR1 control versus theconcentration of the protein tested. 20-fold more SCR1 is required toachieve 50% inhibition than DAF-CR1BB. FIG. 17 shows the percentinhibition of hemolysis of DAF-MCP and DAF control versus theconcentration of the protein tested. To achieve 25 percent inhibition,DAF requires more than 10 fold greater concentration than DAF-MCP.

The results of classical pathway C3 convertase hemolytic assays, usingthe hybrid proteins of the present invention are shown in FIGS. 18A andB, which are graphs showing the percent inhibition of hemolysis versusthe concentration of proteins tested. Again, the hybrid proteincontaining a functional unit of DAF and two functional units of CR1exhibited greater inhibition of hemolysis than protein containing onlyDAF CCPs 1-4.

The ability of the hybrid proteins to inhibit hemolysis in a classicalpathway C5 convertase assay is shown in the graphs of FIGS. 19 and 20.In FIG. 19, DAF-CR1B provides superior inhibition of hemolysis comparedto DAF. In FIG. 20, DAF-CR1BB shows 19-fold greater inhibition ofhemolysis than CR1 and 71-fold greater inhibition than DAF-CR1B.

The ability of DAF-MCP and DAF-CR1BB to act as cofactors for factor I isshown in FIG. 21. Samples were separated by SDS-PAGE, and developed withan anti-human C3 polyclonal antibody. Supernatants from COS cells wereanalyzed neat, with DAF-MCP or with DAF-CR1BB. Each of the samples wasassayed with or without factor I (+I). Supernatants with factor I andeither DAF-MCP or DAF-CR1BB both exhibited 43 and 40 kDa bandscorresponding to formation of iC3b and C3c, respectively. The DAF-CR1BBsample additionally displayed a 29 kDa band corresponding to formationof C3c. A similar result is shown in FIG. 22, where DAF-CR1B acts ascofactor for factor I as shown by the appearance of a band at 29 kDa.

Based upon the foregoing disclosure, it should be apparent that thepresent invention will carry out the aspects set forth above. It istherefore, to be understood that any variations evident fall within thescope of the invention and thus, the selection of specific componentelements can be determined without departing from the spirit of theinvention herein disclosed and described.

1. A protein comprising: a first functional unit of a first complementregulatory protein, wherein the first functional unit exhibitscomplement-regulating properties; a first spacer sequence of at leastabout 200 amino acids encoding a polypeptide that does not exhibitcomplement regulating properties, attached to the first functional unit;and a second functional unit attached to the spacer sequence, selectedfrom the group consisting of polypeptides providing a functional unit ofa second complement regulatory protein, polypeptides derived from animmunoglobulin, and polypeptides that enhance binding of the protein toan animal cell.
 2. The protein of claim 1, additionally comprising asecond spacer sequence of at least about 200 amino acids encoding apolypeptide that does not exhibit complement regulating propertiesattached to the second function domain, and a third functional unitattached to the second spacer, wherein the third functional unit isselected from the group consisting of polypeptides derived from animmunoglobulin, and polypeptides that enhance binding of the protein toan animal cell.
 3. The protein of claim 1, wherein the first functionalunit comprises at least CCPs 2, 3 and 4 of DAF.
 4. The protein of claim1, wherein the second functional unit is selected from the groupconsisting of CCPs 8-10 of Complement Receptor 1 (CR1), CCPs 15-17 ofCR1, CCPs 1-4 of Membrane Cofactor Protein (MCP), polypeptides derivedfrom IgG4, and a lipid tail.
 5. The protein of any of claims 1-4,wherein the spacers are selected from the group consisting ofsubstantially all of the amino acids of CCPs 4-7 of CR1, andsubstantially all of the amino acids of CCPs 11-14 of CR1.
 6. Theprotein of claim 1, wherein the first functional unit comprises CCPs 1,2, 3 and 4 of DAF, the second functional unit is selected from the groupconsisting of CCPs 8-10 of CR1, CCPs 1-4 of Membrane Cofactor Protein(MCP), and polypeptides derived from IgG4, and the first spacer issubstantially all of the amino acids of CCPs 4-7 of CR1.
 7. The proteinof claim 6, additionally comprising a second spacer comprisingsubstantially all of the amino acids of CCPs 4-5 of CR1, and a thirdfunctional unit selected from the group consisting of CCPs 8-10 of CR1CCPs 1-4 of MCP, and polypeptides derived from IgG4.
 8. A polynucleotideencoding the protein of claim
 6. 9. A polynucleotide encoding theprotein of claim
 7. 10. A polynucleotide encoding the protein ofclaim
 1. 11. A vector comprising the polynucleotide of claim
 10. 12. Aprotein having at least 95 percent sequence homology to a proteinselected from the group consisting of proteins having the sequence ofSEQ. ID NO: 13, SEQ. ID NO: 15, SEQ. ID NO: 19, and SEQ. ID NO:
 23. 13.A polynucleotide encoding the protein of claim
 11. 14. A method ofregulating complement activity comprising administering an effectiveamount of protein of claim 1 to a mammal.
 15. The method of claim 15,wherein the mammal is a human.